DNA vaccination, the administration of antigen encoded DNA, is gaining increasing attention as an emerging therapeutic approach for the treatment of many complex disorders including cancer, infectious disease, and allergies (Ishii, K. J. et al. Nature 2008; 451:725-729, Rice, J. et al. Nat. Rev. Cancer. 2008; 8:108-120). DNA vaccines are capable of inducing both humoral and cellular immune responses and are regarded as potentially safer than their attenuated virus counterparts (Gurunathan, S. et al. Annu. Rev. Immunol. 2000; 18:927-974, Liu, M. A. J. Int. Med. 2003; 253:402-410). However, clinical trials have revealed that the immune response induced by a topical injection of naked DNA is insufficient (Roy, M. J. et al. Vaccine, 2000; 19:764-778, Rosenberg, S. A. et al. Hum. Gene Ther. 2003; 14:709-714). Studies have shown that transfection and subsequent activation of antigen presenting cells (APCs) such as dendritic cells (DC) and macrophages are key events in the development of immunity following genetic immunization (Akbari, O. et al. J. Exp. Med. 1999; 189:169-178, Chattergon, M. A. et al. J. Immunol. 1998; 160: 5707-5718). Mountain and co-workers demonstrated that immunization of mice with monocyte-derived dendritic cells transfected with a complex of cationic peptide and a gene encoding tumor associated antigens protected the mice from a lethal challenge with melanoma cells (Irvine, A. S. et al. Nat. Biotechnol. 2000; 18:1273-1278).
Antigen presenting cells such as dendritic cells and macrophages process the antigenic protein through their proteasome complexes into small peptide fragments. These small peptide fragments are then presented to the immune cells (CD8+ and CD4+ T cells) via MHC class I and MHC class II molecules resulting in the induction of cytotoxic T lymphocyte (CTL) and humoral responses (Steinman, R. M. Annu. Rev. Immunol. 1991; 9: 271-296, Banchereau, R. M. and Steinman, R. M. Nature 1998; 392:245-252, Germain, R. N. Cell 1994; 76:287-299, Akbari, O. et al. J. Exp. Med. 1999; 189:169-178, Chattergon, M. A. et al. J. Immunol. 1998; 160:5707-5718, Banchereau, J. and Steinman, R. M. Nature 1998; 392: 245-252). However, antigen presenting cells are hard to transfect. Use of cationic microparticles (Hedley, M. L. et al. Nat. Med. 1998; 4:365-368; Singh, M. et al. Proc. Natl. Acad. Sci. USA 2000; 97:811-816), cationic liposomes (Perrie, Y. et al. Vaccine 2001; 19:3301-3310), and cationic peptide (Irvine, A. S. et al. Nat Biotechnol 2000; 18:1273-1278), etc. have previously been reported for transfection of APCs in ex-vivo. Attempts have been made to increase the potency of immune response through direct transfection of APCs by delivering the antigen encoding DNA via cationic liposomes (Gregoriadis, G. et al. FEBS Lett. 1997; 402:107-110, Klavinskis, L. S. et al. Vaccine 1997; 15: 818-820, Perrie, Y. et al. Vaccine 2001; 19:3301-3310, Hattori, y. et al. Biochem. Biophys. Res. Comm. 2004; 317:992-999). Cationic liposomes owing to their non-toxic and bio-compatible nature offer great advantage over other means of DNA delivery.
A promising approach for enhancing the efficacy of DNA vaccination is based on targeting DNA vaccines to APCs via mannose receptor, a 180 kDa multi-domains unique transmembrane receptors expressed on their cell surfaces (Sallusto, F. et al. J. Exp. Med. 1995; 182:389-400). Previously Srinivas, R. et al. demonstrated that cationic amphiphiles with mannose-mimicking quinic and shikimic acid head-groups can target DNA to antigen presenting cells via mannose receptors (Srinivas, R. et al. J. Med. Chem. 2010; 53:1387-1391). In the same work it was demonstrated that immunization with autologous DCs pre-transfected with electrostatic complexes (lipoplexes) of a plasmid DNA encoding melanoma tumor associated antigen (MART1) and liposomes of two novel amphiphiles with mannose-mimicking quinic and shikimic acid head-groups provides significant protective immunity against lethal melanoma tumor challenge in immunized syngeneic mice (Srinivas, R. et al. J. Med. Chem. 2010; 53:1387-1391). More recently, Srinivas, R. et al. has developed mannose receptor specific lysinylated cationic amphiphiles with mannose-mimicking shikimic and quinic acid head-groups for use in dendritic cell based genetic immunization (Srinivas, R. et al. Indian Patent Application. No. 2170/DEL/2010). However, there a number of time-consuming and cost-ineffective steps to be followed in dendritic cell based genetic immunization processes. One needs to painstakingly isolate the autologous dendritic cells (DCs) from the recipients. The isolated DCs then needs to be ex vivo (outside the body) transfected with DNA vaccines of interest and finally the ex-vivo transfected DCs needs to be re-implanted back into recipient body. Stated differently, the currently practiced ex vivo dendritic cell transfection based genetic immunization procedures are labor-intensive and are likely to be prohibitibly costly for large scale applications. To this end, using electroporation technique for delivering DNA, Steinman and coworkers succeeded in enhancing the efficacy of genetic immunization by targeting DNA vaccines to DCs under in-vivo settings. Their approach is based on construction of DNA vaccine encoding antigenic protein and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205 (Nchinda, G. et al. J. Clin. Invest. 2008; 118:1427-1436; Nchinda, G. et al. Proc. Natl Acad. Sci. USA. 2010; 107: 4281). However, large scale construction of such DNA vaccines encoding both antigenic proteins and scFv is unlikely to be cost-effective. More recently Hashida and coworkers reported development of mannose-receptor selective and ultrasound-responsive mannosylated liposomes for in vivo transduction of DCs in genetic immunization (Un K. et al. Biomaterials 2010; 31: 7813-7826; Un K. et al. Mol Pharm 2011; 8: 543-554).
Using p-CMV-β-gal (encoding β-galactosidase enzyme) as a model DNA vaccine, the present invention discloses that direct in vivo administration (i.e. without the need of isolating autologous DCs) of the electrostatic complexes of p-CMV-β-gal and liposomes of the presently described mannose-receptor selective lysinylated cationic amphiphiles containing both guanidine and mannose-mimicking shikimoyl head-groups in mice are highly efficient in eliciting both cellular and humoral immune responses against β-gal antigen. This invention also discloses the applications of the presently described lysinylated cationic amphiphiles with both guanidine and mannose-mimicking shikimoyl head-group in genetic immunization using DNA vaccines encoding Gp100 and tyrosinase, two human melanocyte lineage-specific antigens expressed by majority of human malignant melanoma (Coulie P. G. et al. J Exp Med 1994; 180: 35-42; Kawakami Y. et al. Proc Natl Acad Sci USA 1994; 91: 3515-9; Topalian S. L. et al. Proc Natl Acad Sci USA. 1994; 91: 9461-9465; Brichard V. et al. J. Exp Med. 1993; 178: 489-95). These antigens share 77% & 82% amino-acid sequence identities, respectively, with their murine counterparts (Zhai Y et al. J Immunother 1997; 20: 15-25; Colella A. T. et al. J. Exp Med. 2000; 191: 1221-1231). The present invention discloses that direct in vivo immunization with electrostatic complexes (lipoplexes) of DNA vaccines p-CMV-gp100 and p-CMV-tyrosinase (encoding melanoma antigens gp-100 & tyrosinase, respectively) and liposome of the presently described lysinylated cationic amphiphiles with both guanidine and mannose-mimicking shikimic acid head-groups provides long-lasting (100 days post tumor challenge) tumor protection against aggressive melanoma tumor challenge in immunized mice. The presently described simple non-viral in vivo DC-targating system may find future exploitations in inducing long-lasting immune response in genetic immunization.